Methods of using combinations of siRNAs for treating a disease or a disorder, and for enhancing siRNA efficacy in RNAi

Abstract

The present invention provides methods for treating diseases or disorders, and methods for enhancing siRNA efficacy in RNAi, including administering to a subject or a biological system one or more siRNAs capable of down regulating the expression of one or more target genes and one or more siRNAs capable of down regulating the expression of one or more negative regulators of RNAi. The present invention also provides compositions including one or more siRNAs, or precursors thereof, capable of down regulating the expression of one or more target genes and comprising one or more siRNAs, or precursors thereof, capable of down regulating the expression of one or more negative regulators of RNAi.

1 . A method for treating a disease or a disorder, the method comprising administering to a subject (i) one or more siRNAs capable of down regulating expression of one or more target genes, and (ii) one or more siRNAs capable of down regulating expression of one or more negative regulators of RNAi. 
     
     
         2 . The method of  claim 1 , wherein the ratio of the siRNAs capable of down regulating expression of target genes to the siRNAs capable of down regulating expression of negative regulators of RNAi is in a range of about 5:1 to about 20:1 (w/w). 
     
     
         3 . The method of  claim 2 , wherein the ratio of the siRNAs capable of down regulating expression of target genes to the siRNAs capable of down regulating expression of negative regulators of RNAi is about 10:1 (w/w). 
     
     
         4 . The method of  claim 1 , wherein the siRNAs capable of down regulating expression of target genes and the siRNAs capable of down regulating expression of negative regulators of RNAi are administered at the same time. 
     
     
         5 . The method of  claim 1 , wherein the siRNAs capable of down regulating expression of target genes are administered after the siRNAs capable of down regulating expression of negative regulators of RNAi have been administered, and still retain their activity. 
     
     
         6 . The method of  claim 5 , wherein the siRNAs capable of down regulating expression of target genes are administered within 3 days after administration of the siRNAs capable of down regulating expression of negative regulators of RNAi. 
     
     
         7 . The method of  claim 1 , wherein the siRNAs capable of down regulating expression of negative regulators of RNAi are administered after the siRNAs capable of down regulating expression of target genes have been administered, and still retain their activity. 
     
     
         8 . The method of  claim 7 , wherein the siRNAs capable of down regulating expression of negative regulators of RNAi are administered within 3 days after administration of the siRNAs capable of down regulating expression of target genes. 
     
     
         9 . The method of  claim 1 , wherein all or a portion of the siRNAs are chemically synthesized. 
     
     
         10 . The method of  claim 1 , wherein all or a portion of the siRNAs are synthesized in vivo or in vitro using a nucleic acid sequence. 
     
     
         11 . The method of  claim 1 , wherein all or a portion of the siRNAs are derived in vivo or in vitro from precursor RNAs via chemical modification, biological modification or combinations thereof. 
     
     
         12 . The method of  claim 1 , wherein the disease is a cancer. 
     
     
         13 . The method of  claim 12 , wherein the cancer is selected from the group consisting of pancreatic carcinoma, melanoma, colon carcinoma, lung carcinoma, kidney carcinoma, gastrointestinal stromal tumors (GIST), chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), breast cancer, glioblastoma, ovarian carcinoma, endometrial carcinoma, hepatocellular carcinoma, renal cell carcinoma, thyroid carcinoma, lymphoid carcinoma, bladder carcinoma, prostate carcinoma, cervical carcinoma, non-Hodgkin lymphoma, oral cavity & pharynx carcinoma, head and neck cell carcinoma, stomach carcinoma, esophagus carcinoma, larynx carcinoma, brain & ONS carcinoma, liver & IBD carcinoma, ovarian carcinoma, and nasopharyngeal carcinoma. 
     
     
         14 . The method of  claim 13 , wherein the cancer is a melanoma. 
     
     
         15 . The method of  claim 1 , wherein the disease is a disease caused by a virus. 
     
     
         16 . The method of  claim 15 , wherein the disease is selected from the group consisting of acquired immunodeficiency syndrome (AIDS), hepatitis A, hepatitis B, hepatitis C, hepatitis Delta, influenza, foot-and-mouth disease, dengue disease/hemorrhagic disease, measles/subacute sclerosing panencephalitis (SSPE), cephalitis and brain infection, glandular fever/chronic lymphocytic leukemia/lymphomas/nasopharyngeal carcinoma, adult T cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a neurologic disease, cytomegalovirus inclusion disease/transplant arterial disease, sexually transmitted infection (STI), oral and cervical cancer/head and neck cancer/squamous cell carcinoma, fever blisters, genital sores and a flu-like illness. 
     
     
         17 . The method of  claim 16 , wherein the disease is hepatitis B. 
     
     
         18 . The method of  claim 1 , wherein the target gene is a gene associated with a disease, whose down regulation ameliorates the disease. 
     
     
         19 . The method of  claim 18 , wherein the target gene is a gene encoding a product selected from the group consisting of VEGF, VEGFR, c-Raf/bcl-2, CEACAM6, EGFR, Bcr-abl, AML1/MTG8, Btk, LPA1, Csk, PKC-theta, Bim1, P53 mutant, stat3, c-myc, SIRT1, ERK1, Cyclooxygenase-2, sphingosine 1-phosphate (SIP) receptor-1, insulin-like growth factor receptor, Bax, CXCR4, FAK, EphA2, Matrix metalloproteinase, BRAF(V599E), Brk, EBV, FASE, C-erbB-2/HER2, HPV E6\E7, Livin/ML-LAP/KIAP, MDR, CDK-2, MDM-2, PKC-α, TGF-β, H-Ras, K-Ras, PLK1, Telomerase, S100A10, NPM-ALK, Nox1, Cyclin E, Gp210, c-Kit, survivin, Philadelphia chromosome, Ribonucleotide reductase, Rho C, ATF2, P110a, P110B of PI 3 kinase, Wt1, Pax2, Wnt4, beta-catanin, integrin, urokinase-type plasminogen activator, Hec1, Cyclophilin A, DNMT, MUC, Acetyl-CoA Carboxylase {alpha}, Mirk/Dyrk1b, MTA1, SMYD3, ACTR, Hath1, Mad2, STK15, XIAP, CD147/EMMPRIN, ENPP2/ATX /ATX-X/FLJ26803/LysoPLD/NPP2/PD-IALPHA/PDNP2, AKT, PrPC, thioredoxin reductase 1, HSPG2, p38 MAP kinase, hTERT, alphaB-Crystallin, STAT6, choline kinase, cyclin D1/CDK4, ASH1, osteopontin, 3-alkyladenine-DNA glycosylase, Plasmalemmal vesicle associated protein-1, SHP2, STAT5, Gab2, Etk/BMX, AFP, Id1/Id3 gene, Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38, phosphatidylethanolamine-binding protein 4, ATP citrate lyase, cyclophilin A, DNA-PK, CT120A, EBNA1, Pim family kinases, hypoxia-inducible factor-1 alpha, acetyl-CoA-carboxylase-alpha, Rac 1/RAC3, Aurora-B, platelet-derived growth factor-D/platelet-derived growth factor receptor beta, Androgen Receptor, EN2, Vav1, BRCA1, Pyk2, leptin, hLRH-1, p28GANK, MCT-1, Fibroblast growth factor receptor 3, p53R2, integrin-linked kinase, cdc42, MAT2A, ICAMs, mimitin, RET, S-phase kinase-interacting protein 2, NRAS, phosphatidylinositol 3-kinase, Fas-ligand, IGFBP-5, E2F4, FLT3, estrogen receptor, LYN kinase, cathepsin B, ZNRD1, ARA55 and activin. 
     
     
         20 . The method of  claim 19 , wherein the target gene is the c-myc gene. 
     
     
         21 . The method of  claim 1 , wherein the target gene is a viral gene. 
     
     
         22 . The method of  claim 21 , wherein the target gene is a gene of a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis delta virus, influenza virus, foot-and-mouth disease virus, dengue virus type 2, measles virus, encephalitis virus, Epstein-Barr virus, human T-cell leukemia virus, cytomegalovirus, human papillomavirus and herpes simplex virus. 
     
     
         23 . The method of  claim 22 , wherein the target gene is a gene encoding the polymerase of hepatitis B virus. 
     
     
         24 . The method of  claim 1 , wherein the negative regulators of RNAi are selected from the group consisting of exonucleases and adenosine deaminases. 
     
     
         25 . The method of  claim 24 , wherein the exonuclease is THEX1 or a homolog thereof. 
     
     
         26 . The method of  claim 24 , wherein the adenosine deaminase is ADAR1 or a homolog thereof. 
     
     
         27 . The method of  claim 1 , wherein the target gene is a c-myc gene and the negative regulator of RNAi is THEX1, ADAR1, or a combination thereof. 
     
     
         28 . The method of  claim 1 , wherein the target gene is a gene encoding the polymerase of hepatitis B virus and the negative regulator of RNAi is THEX1. 
     
     
         29 . A method of enhancing siRNA efficacy, the method comprising administering to a biological system (i) one or more siRNAs capable of down regulating expression of one or more target genes, and (ii) one or more siRNAs capable of down regulating expression of one or more negative regulators of RNAi. 
     
     
         30 . The method of  claim 29 , wherein the ratio of the siRNAs capable of down regulating expression of target genes to the siRNAs capable of down regulating expression of negative regulators of RNAi is in a range of about 5:1 to about 20:1 (w/w). 
     
     
         31 . The method of  claim 30 , wherein the ratio of the siRNAs capable of down regulating expression of target genes to the siRNAs capable of down regulating expression of negative regulators of RNAi is about 10:1 (w/w). 
     
     
         32 . The method of  claim 29 , wherein the siRNAs capable of down regulating expression of target genes and the siRNAs capable of down regulating expression of negative regulators of RNAi are administered at the same time. 
     
     
         33 . The method of  claim 29 , wherein the siRNAs capable of down regulating expression of target genes are administered after the siRNAs capable of down regulating expression of negative regulators of RNAi have been administered, and still retain their activity. 
     
     
         34 . The method of  claim 33 , wherein the siRNAs capable of down regulating expression of target genes are administered within 3 days after administration of the siRNAs capable of down regulating expression of negative regulators of RNAi. 
     
     
         35 . The method of  claim 29 , wherein the siRNAs capable of down regulating expression of negative regulators of RNAi are administered after the siRNAs capable of down regulating expression of target genes have been administered, and still retain their activity. 
     
     
         36 . The method of  claim 35 , wherein the siRNAs capable of down regulating the expression of negative regulators of RNAi are administered within 3 days after administration of the siRNAs capable of down regulating the expression of target genes. 
     
     
         37 . The method of  claim 29 , wherein all or a portion of the siRNAs are chemically synthesized. 
     
     
         38 . The method of  claim 29 , wherein all or a portion of the siRNAs are synthesized in vivo or in vitro using a nucleic acid sequence. 
     
     
         39 . The method of  claim 29 , wherein all or a portion of the siRNAs are derived in vivo or in vitro from precursor RNAs via chemical modification, biological modification, or combination thereof. 
     
     
         40 . The method of  claim 29 , wherein the target gene is a gene associated with a disease, whose down regulation ameliorates the disease. 
     
     
         41 . The method of  claim 40 , wherein the target gene is a gene encoding a product selected from the group consisting of VEGF, VEGFR, c-Raf/bcl-2, CEACAM6, EGFR, Bcr-abl, AML1/MTG8, Btk, LPA1, Csk, PKC-theta, Bim1, P53 mutant, stat3, c-myc, SIRT1, ERK1, Cyclooxygenase-2, sphingosine 1-phosphate (SIP) receptor-1, insulin-like growth factor receptor, Bax, CXCR4, FAK, EphA2, Matrix metalloproteinase, BRAF(V599E), Brk, EBV, FASE, C-erbB-2/HER2, HPV E6\E7, Livin/ML-IAP/KIAP, MDR, CDK-2, MDM-2, PKC-α, TGF-β, H-Ras, K-Ras, PLK1, Telomerase, S100A10, NPM-ALK, Nox1, Cyclin E, Gp210, c-Kit, survivin, Philadelphia chromosome, Ribonucleotide reductase, Rho C, ATF2, P110a, P110B of PI 3 kinase, Wt1, Pax2, Wnt4, beta-catanin, integrin, urokinase-type plasminogen activator, Hec1, Cyclophilin A, DNMT, MUC1, Acetyl-CoA Carboxylase {alpha}, Mirk/Dyrk1b, MTA1, SMYD3, ACTR, Hath1, Mad2, STK15, XIAP, CD147/EMMPRIN, ENPP2/ATX /ATX-X/FLJ26803/LysoPLD/NPP2/PD-IALPHA/PDNP2, AKT, PrPC, thioredoxin reductase 1, HSPG2, p38 MAP kinase, hTERT, alphaB-Crystallin, STAT6, choline kinase, cyclin D1/CDK4, ASH1, osteopontin, 3-alkyladenine-DNA glycosylase, Plasmalemmal vesicle associated protein-1, SHP2, STAT5, Gab2, Etk/BMX, AFP, Id1/Id3 gene, Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38, phosphatidylethanolamine-binding protein 4, ATP citrate lyase, cyclophilin A, DNA-PK, CT120A, EBNA1, Pim family kinases, hypoxia-inducible factor-1alpha, acetyl-CoA-carboxylase-alpha, Rac1/RAC3, Aurora-B, platelet-derived growth factor-D/platelet-derived growth factor receptor beta, Androgen Receptor, EN2, Vav1, BRCA1, Pyk2, leptin, hLRH-1, p28GANK, MCT-1, Fibroblast growth factor receptor 3, p53R2, integrin-linked kinase, cdc42, MAT2A, ICAMs, mimitin, RET, S-phase kinase-interacting protein 2, NRAS, phosphatidylinositol 3-kinase, Fas-ligand, IGFBP-5, E2F4, FLT3, estrogen receptor, LYN kinase, cathepsin B, ZNRD1, ARA55 and activin. 
     
     
         42 . The method of  claim 41 , wherein the target gene is the c-myc gene. 
     
     
         43 . The method of  claim 29 , wherein the target gene is a viral gene. 
     
     
         44 . The method of  claim 43 , wherein the target gene is a gene of a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis delta virus, influenza virus, foot-and-mouth disease virus, dengue virus type 2, measles virus, encephalitis virus, Epstein-Barr virus, human T-cell leukemia virus, cytomegalovirus, human papillomavirus and herpes simplex virus. 
     
     
         45 . The method of  claim 44 , wherein the target gene is a gene encoding the polymerase of hepatitis B virus. 
     
     
         46 . The method of  claim 29 , wherein the negative regulators of RNAi are selected from the group consisting of exonucleases and adenosine deaminases. 
     
     
         47 . The method of  claim 46 , wherein the exonuclease is THEX1 or a homolog thereof. 
     
     
         48 . The method of  claim 46 , wherein the adenosine deaminase is ADAR1 or a homolog thereof. 
     
     
         49 . The method of  claim 29 , wherein the target gene is the c-myc gene and the negative regulator of RNAi is THEX1. 
     
     
         50 . The method of  claim 29 , wherein the target gene is the gene encoding polymerase of hepatitis B virus and the negative regulator of RNAi is THEX1. 
     
     
         51 . A composition comprising one or more siRNAs, or precursors thereof, capable of down regulating expression of one or more target genes and comprising one or more siRNAs, or precursors thereof, capable of down regulating expression of one or more negative regulators of RNAi. 
     
     
         52 . A composition comprising one or more nucleotide sequences encoding one or more siRNAs, or precursors thereof, capable of down regulating expression of one or more target genes and comprising one or more siRNAs, or precursors thereof, capable of down regulating expression of one or more negative regulators of RNAi. 
     
     
         53 . A composition comprising one or more siRNAs, or precursors thereof, capable of down regulating expression of one or more target genes and comprising one or more nucleotide sequences encoding one or more siRNAs, or precursors thereof, capable of down regulating expression of one or more negative regulators of RNAi. 
     
     
         54 . A composition comprising one or more nucleotide sequences encoding one or more nucleotide sequences encoding one or more siRNAs, or precursors thereof, capable of down regulating expression of one or more target genes and comprising one or more nucleotide sequences encoding one or more siRNAs, or precursors thereof, capable of down regulating expression of one or more negative regulators of RNAi. 
     
     
         55 . The composition of  claim 51 , wherein the ratio of the siRNAs capable of down regulating expression of target genes to the siRNAs capable of down regulating expression of negative regulators of RNAi is in a range of about 5:1 to about 20:1 (w/w) 
     
     
         56 . The composition of  claim 51 , wherein the ratio of the siRNAs capable of down regulating expression of target genes to the siRNAs capable of down regulating expression of negative regulators of RNAi is about 10:1 (w/w). 
     
     
         57 . The composition of  claim 51 , wherein all or a portion of the siRNAs are chemically synthesized. 
     
     
         58 . The composition of  claim 51 , wherein all or a portion of the siRNAs are synthesized in vivo or in vitro using a nucleic acid sequence. 
     
     
         59 . The composition of  claim 51 , wherein the target gene is a gene associated with a disease, whose down regulation ameliorates the disease. 
     
     
         60 . The composition of  claim 59 , wherein the target gene is a gene encoding a product selected from the group consisting of VEGF, VEGFR, c-Raf/bcl-2, CEACAM6, EGFR, Bcr-abl, AML1/MTG8, Btk, LPA1, Csk, PKC-theta, Bim1, P53 mutant, stat3, c-myc, SIRT1, ERK1, Cyclooxygenase-2, sphingosine 1-phosphate (SIP) receptor-1, insulin-like growth factor receptor, Bax, CXCR4, FAK, EphA2, Matrix metalloproteinase, BRAF(V599E), Brk, EBV, FASE, C-erbB-2/HER2, HPV E6\E7, Livin/ML-IAP/KIAP, MDR, CDK-2, MDM-2, PKC-α, TGF-β, H-Ras, K-Ras, PLK1, Telomerase, S100A10, NPM-ALK, Nox1, Cyclin E, Gp210, c-Kit, survivin, Philadelphia chromosome, Ribonucleotide reductase, Rho C, ATF2, P110a, P110B of PI 3 kinase, Wt1, Pax2, Wnt4, beta-catanin, integrin, urokinase-type plasminogen activator, Hec1, Cyclophilin A, DNMT, MUC1, Acetyl-CoA Carboxylase {alpha}, Mirk/Dyrk1b, MTA1, SMYD3, ACTR, Hath1, Mad2, STK15, XIAP, CD147/EMMPRIN, ENPP2/ATX /ATX-X/FLJ26803/LysoPLD/NPP2/PD-IALPHA/PDNP2, AKT, PrPC, thioredoxin reductase 1, HSPG2, p38 MAP kinase, hTERT, alphaB-Crystallin, STAT6, choline kinase, cyclin D1/CDK4, ASH1, osteopontin, 3-alkyladenine-DNA glycosylase, Plasmalemmal vesicle associated protein-1, SHP2, STAT5, Gab2, Etk/BMX, AFP, Id1/Id3 gene, Maternal embryonic leucine zipper kinase/murine protein serine-threonine kinase 38, phosphatidylethanolamine-binding protein 4, ATP citrate lyase, cyclophilin A, DNA-PK, CT120A, EBNA1, Pim family kinases, hypoxia-inducible factor-1 alpha, acetyl-CoA-carboxylase-alpha, Rac1/RAC3, Aurora-B, platelet-derived growth factor-D/platelet-derived growth factor receptor beta, Androgen Receptor, EN2, Vav1, BRCA1, Pyk2, leptin, hLRH-1, p28GANK, MCT-1, Fibroblast growth factor receptor 3, p53R2, integrin-linked kinase, cdc42, MAT2A, ICAMs, mimitin, RET, S-phase kinase-interacting protein 2, NRAS, phosphatidylinositol 3-kinase, Fas-ligand, IGFBP-5, E2F4, FLT3, estrogen receptor, LYN kinase, cathepsin B, ZNRD1, ARA55 and activin. 
     
     
         61 . The composition of  claim 60 , wherein the target gene is the c-myc gene. 
     
     
         62 . The composition of  claim 51 , wherein the target gene is a viral gene. 
     
     
         63 . The composition of  claim 62 , wherein the target gene is a gene of a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis delta virus, influenza virus, foot-and-mouth disease virus, dengue virus type 2, measles virus, encephalitis virus, Epstein-Barr virus, human T-cell leukemia virus, cytomegalovirus, human papillomavirus and herpes simplex virus. 
     
     
         64 . The composition of  claim 63 , wherein the target gene is a gene encoding the polymerase of hepatitis B virus. 
     
     
         65 . The composition of  claim 51 , wherein the negative regulators of RNAi are selected from the group consisting of exonucleases and adenosine deaminases. 
     
     
         66 . The composition of  claim 65 , wherein the exonuclease is THEX1 or a homolog thereof. 
     
     
         67 . The composition of  claim 65 , wherein the adenosine deaminase is ADAR1 or a homolog thereof. 
     
     
         68 . The composition of  claim 51 , wherein the target gene is the c-myc gene and the negative regulator of RNAi is THEX1. 
     
     
         69 . The composition of  claim 51 , wherein the target gene is the gene encoding polymerase of hepatitis B virus and the negative regulator of RNAi is THEX1. 
     
     
         70 . A method of determining an optimal ratio of siRNAs capable of down regulating expression of one or more target genes to siRNAs capable of down regulating expression of one or more negative regulators of RNAi in methods for treating a disease or a disorder and in methods for enhancing siRNA efficacy, comprising the following steps:
 (a) inducing expression of genes encoding a negative regulator of RNAi using any siRNA molecules;   (b) determining an effective dose of siRNA molecules that is able to induce high expression of the negative regulator of RNAi;   (c) based on the high expression of the negative regulator of RNAi determined in (b), determining a dose of an siRNA that down regulates expression of the negative regulator of RNAi to a base expression level; and   (d) based on the down regulation of the negative regulator of RNAi determined in (c), determining a dose of an siRNA that down regulates expression of one or more target genes.